Mg2+ translocation across cellular membranes is crucial for
a myriad of physiological processes. Eukaryotic Mrs2 transporters
are distantly related to the major bacterial Mg2+
transporter CorA, the structure of which displays a bundle
of giant -helices forming a long pore that extends beyond
the membrane before widening into a funnel-shaped cytosolic
domain. Here, a functional and structural analysis of the
regulatory domain of the eukaryotic Mg2+ channel Mrs2 from
the yeast inner mitochondrial membrane is presented using
crystallography, genetics, biochemistry and fluorescence
spectroscopy. Surprisingly, the fold of the Mrs2 regulatory
domain bears notable differences compared with the related
bacterial channel CorA. Nevertheless, structural analysis
showed that analogous residues form functionally critical
sites, notably the hydrophobic gate and the Mg2+-sensing site.
Validation of candidate residues was performed by functional
studies of mutants in isolated yeast mitochondria. Measurements
of the Mg2+ influx into mitochondria confirmed the
involvement of Met309 as the major gating residue in Mrs2,
corresponding to Met291 in CorA.

Mg2+ translocation across cellular membranes is crucial for
a myriad of physiological processes. Eukaryotic Mrs2 transporters
are distantly related to the major bacterial Mg2+
transporter CorA, the structure of which displays a bundle
of giant -helices forming a long pore that extends beyond
the membrane before widening into a funnel-shaped cytosolic
domain. Here, a functional and structural analysis of the
regulatory domain of the eukaryotic Mg2+ channel Mrs2 from
the yeast inner mitochondrial membrane is presented using
crystallography, genetics, biochemistry and fluorescence
spectroscopy. Surprisingly, the fold of the Mrs2 regulatory
domain bears notable differences compared with the related
bacterial channel CorA. Nevertheless, structural analysis
showed that analogous residues form functionally critical
sites, notably the hydrophobic gate and the Mg2+-sensing site.
Validation of candidate residues was performed by functional
studies of mutants in isolated yeast mitochondria. Measurements
of the Mg2+ influx into mitochondria confirmed the
involvement of Met309 as the major gating residue in Mrs2,
corresponding to Met291 in CorA.